Calcified nodules in control group (200). fThe calcium nodules of experimental group. the mRNA expressions of collagen I and osteocalcin were detected by MTT and RT-PCR assays, respectively and alkaline phosphatase(ALP)and calcium concentration at different induction time were detected. The cell proliferation curves were S shaped. The OD values of experimental group were higher than those of control group at 1, three or more, 5, 7, 14, and 21 days after osteogenic induction (P < 0. 05). ALP and alizarin red stains of ADSCs were all positive, but golden round nodes became bigger and more in the experimental group compared with the control group after 2 weeks. At 7 and 14 days, collagen I and osteocalcin mRNA expression were greater in the experimental group than the control group. ALP and calcium concentration of experimental group were higher than that of control group at 1, 2, 3, 4 weeks after osteogenic induction (P < 0. 05). Thus, these results show that the CGRP-induced ADSCs combined with calcium alginate gel to osteoblasts differentiation. Keywords: Calcitonin gene-related peptide, Adipose-derived stem cells, Osteogenic differentiation, Calcium alginate gel, Three-dimensional cultures == Introduction == Calcitonin gene-related peptide (CGRP) is one of the most abundant neuropeptides. So far CGRP research has concentrated on its role around the nervous and the cardiovascular systems [13], but not on bone defect repair. Recent studies discovered that CGRP also plays an important role in bone repair and bone reconstruction. Hukkanen et al. (1993) found that CGRP-positive nerve fibers initially show degradation of periosteum and then quickly proliferate around the bone fracture in rats [4]. It is followed by the progress of the callus formation and bone remodeling along with the change in distribution JC-1 and density. Aoki et al. (2004) verified that a large number of CGRP-positive nerve fibers were emerged in fiber granulation tissue, periosteum, and bone tissue during healing process of fracture in rats in an orderly manner with the degree of callus formation and hyperplasia of bone remodeling [5]. These hyperplasia sensory nerve fibers can release higher level of CGRP than the serum to participate in the normal process of bone healing. Li et al. (2007) also found JC-1 that bone fracture can stimulate the generation of a large number of CGRP-positive nerve fibers around the injury in the rat model of tibial fracture JC-1 which perhaps is a prerequisite intended for fracture recovery and remodeling [6]. Hayashi et al. (2009) found that serum CGRP expression in rats markedly increased in early stage of spinal cord injury with fracture and the level is up to three times more than normal after 7 days of injury [7]. The difference between the level of CGRP expression after 3 and 7 days of injury and simple fracture was statistically significant (P < 0. 01). Onuoha (2011) observed a change in plasma CGRP level (detected by ELISA method) within 24 h in patients after bone fracture. The plasma CGRP levels were significantly elevated in patients with bone fracture than the control group [8]. Sun Xiao-xin et al. (2009) reported that the Rabbit polyclonal to ZC3H11A CGRP expression in all cells which participated in the bone healing of patients group with fracture and complications of central nervous system injury was significantly higher than the simple fracture group at each time point in different stages of fracture healing through animal experiments. Also the formation and remodeling of callus at the fracture end happened earlier than that in the simple fracture group [9]. Ekelund et al. (1997) discovered that distribution density of CGRP-positive nerve fibers was positively related JC-1 to the local amount of bone formation based on the study of distribution of the nerve fibers in the heterotopic ossification [10]. The CGRP-positive nerve fibers were sparse or lacking in the nonunion human long bone shaft fracture. Furthermore, removal of sensory nerve terminals of CGRP-positive nerve fibers in the periosteum of rat tibia fracture, led to nonunion of fracture [11]. When the sciatic nerve was cut, the rat tibial fracture callus became bigger than normal but with low density and poor mechanical properties, and the callus grew without CGRP-positive nerve fibers [12]. In the rat model of spinal cord injury, formation of a large number of fibrous callus and.